1. Field of the Invention
This invention relates generally to the subject matter of inorganic and biological CaCO.sub.3 formation. More particularly, it relates to the inhibition of CaCO.sub.3 deposition by a proteinaceous fraction obtained from CaCO.sub.3 -forming organisms. This proteinaceous fraction has been found effective for the inhibition of inorganic or biological CaCO.sub.3 -deposition on a surface with which it is contacted.
2. Description of the Prior Art
Control of CaCO.sub.3 -encrustation and growth of calcifying organisms on surfaces in marine environments has long been recognized as a potentially solvable problem. By preventing or slowing the occurrence of these "fouling" substances in organisms, the useful lifetime of surfaces such as hulls of ships and pilings of docks can be increased. In the case of hulls of ships, prevention of fouling also has the effect of allowing the ship to move more efficiently through the water.
Historically, the problem has been approached by impregnating or coating surfaces with compounds that interfere with the metabolism of fouling organisms. For example, the use of inhibitors of carbonic anhydrase, an enzyme often involved in calcification, has been suggested for such use (Costlow, J. D., Physiological Zoology, 32:177 (1959)). More recently, inhibitors of the enzyme polyphenol oxidase, also involved in the calcification process, have been shown as effective anti-fouling compounds (Turner, R. D., Symposium on Marine Biodeterioration, Naval Institute Press, Washington, D.C.). Less specific metabolic inhibitors, such as organotin compounds, are also being applied (Good, M. L., Symposium on Marine Biodeterioration, supra).
In addition, CaCO.sub.3 crystal growth occurs abiotically in most natural solutions leading to unwanted calcified deposits. For example, scale builds up anywhere in the sea where nucleation occurs, because sea water is supersaturated with respect to CaCO.sub.3 by a factor of 5 to 10-fold, allowing crystal growth to proceed spontaneously (Stumm, W. and Morgan, J. J., Aquatic Chemistry, John Wiley and Sons, Somerset, N.J. (1981)). Inorganic scales are also often encountered as unwanted deposits in pipes and boilers where supersaturation becomes a problem due to evaporative concentrations of ions. Carboxylates, such as NTA, ethylene diamine tetraacetate (EDTA) and gluconates have been used to retard or inhibit the precipitation of supersaturated solutions of calcium carbonate, although somewhat high concentrations are needed for these compounds to act as effective inhibitors. Hexametaphosphate, at 1-10 ppm concentration was found to retard scaling leading to the widespread use of polyphosphates as scale inhibitors in municipal and industrial water systems. (Monsanto's Technical Bulletin No. IC/SCS-323, Dequest 2010 Phosphonate).
In recirculating cooling water systems, calcium carbonate is generally the predominant scalant. Since cooling towers are efficient air scrubbers, this circulating water is saturated with CO.sub.2, establishing an equilibrium between bicarbonate and carbonate in solution. As the pH of the water rises, this equilibrium shifts toward carbonate. Heating also forms a shift in the dissolved inorganic carbon equilibrium to the right, producing calcium carbonate: ##STR1## Finally, calcium carbonate shows an inverse solubility trend, being less soluble at higher temperatures. All of these factors tend to produce scaling on critical heat-transfer surfaces which reduces the heat transfer efficiency, increases frequency of required cleaning and decreases the lift of the system. Several of the inhibitors of the precipitation of calcium carbonate show the phenomenon of a threshold effect, e.g., the prevention of precipitation from supersaturated solutions of scalants by substoichiometric levels of inhibitors. Present mechanistic theories postulate that the threshold agent is adsorbed on the growth sites of the scalant crystallite during the process of crystallization and alters the growth pattern so that the resultant scalant crystals are formed more slowly and are highly distorted. (Reddy M. M. and Nancollas, G. H., Desalination 12:61 (1973)).
A speculative model of organic matrix structure and function, based primarily on aspects of mollusk shell proteinaceous matrix biochemistry, as well as a brief review of the proteinaceous organic matrices from various other phyla was presented by Weiner, S., Traub, W. and Lowenstam, H. A., "Organic Matrix in Calcified Exoskeletons" in Biomineralization and Biological Metal Accum., pp. 205-224 (1983), Westbroek and De Jong, Eds., Reidel Publishing Co. Further characterization of the various matrical components, such as the soluble matrical fraction containing glycoprotein components can be found in Krampitz, G., Drolshagen, H., Hausle, J., and Hof-Irmscher, K, "Organic Matrices of Mollusk Shell", in Biomineral. and Biol. Metal Accum., supra, pp. 231-247 (1983), incorporated herein by reference. Calcium-binding, sulfated, high molecular weight glycoproteins have been identified in the soluble matrix of several species. In addition, this soluble fraction may also contain a number of smaller molecular weight components (Weiner, F. Lowenstam, H. A. and Hood, L. J., J. Exp. Mar. Biol. Ecol., 30:45-51 (1977), incorporated herein by reference). A further characterization of the amino acid sequence of soluble mollusk shell protein by peptide analysis after cleavage of the proteins on both sides of the Asp residues, showed a pattern of a repeating sequence of aspartic acids separated by either glycine or serine in an alternative manner with Asp. The repeating sequence observed is of the form (Asp-Y).sub.n -type, where Y is a single amino acid. The organic matrix of almost all mineralized tissues studied to date (both vertebrates and invertebrates) contain proteins which are enriched in aspartic acid (Asp) and/or glutamic acid (Glu) (Veis, A., and Perry A., Biochemistry 6:2049 (1967)); Shuttleworth, A. and Veis, A., Biochem. Biophys. Acta, 257:414 (1972), incorporated herein by reference).
The (Asp-Y).sub.n -type sequence was hypothesized to be present in the organic matrices from a variety of mollusks species, such as Crassostrea virginica, Mercenaria mercenaria, Crassostrea irredescens and Nautilus pompilius, and suggested that these sequences played a function as a template for mineralization, although X-ray diffraction studies showed that there was a poor match between the Ca to Ca distances in the crystal lattice and the potential calcium-binding sites along the polypeptide chain for this sequence (Weiner S. and Hood L., Science 19: 987 (1975); Weiner S., in The Chem. and Biol. of Mineral. Connective Tissues, Veis A., ed., pp., 517-521, Elsevier North Holland, Inc. (1981); and Weiner S. and Traub W., in Struct. Asp. of Recog. And Assembly in Biol. Mascromolec. Balaban, N., Sussman, J. L., Traub, W. and Yonath, A., Eds., pp. 467-482 (1981), incorporated herein by reference).
Acknowledging that the process of CaCO.sub.3 nucleation and crystal growth itself is central to the process of encrustation by all calcifying organisms, whether they are barnacles, oysters, ship worms, algae and the like, Wheeler, A. P., George, J. W. and Evans, C. A., Science 212: 1397 (1981), incorporated herein by reference, made the discovery that a 170,000 MW glycoprotein obtained from the proteinaceous matrix that permeates the CaCO.sub.3 of oyster shell is a very potent inhibitor, rather than an initiator of CaCO.sub.3 nucleation and crystal growth as previously thought. The 170,000 glycoprotein was identified by staining for carbohydrates and it was shown to contain 10.2% carbohydrate by weight. The molecular weight and carbohydrate content reported for the glycoprotein from oyster shell are comparable to those observed for the protein obtained from clams by Crenshaw, M. A., Biomineralization 6: 6 (1972), incorporated herein by reference. Sikes, C. S. and Wheeler, A. P., in concurrently filed and co-pending U.S. application Ser. No. 563,252 entitled "Inhibition of Inorganic and Biological CaCO.sub.3 Deposition by a Polysaccharide Fraction Obtained From CaCO.sub.3 -Forming Organisms", incorporated herein by reference, further disclose a method of inhibiting the formation of CaCO.sub.3 -containing deposits with a polysaccharide-containing fraction obtained from CaCO.sub.3 -forming organisms. The polysaccharide materials disclosed therein are substantially devoid of protein or polypeptide structures.
Sikes, C. S. and Wheeler, A. P., in concurrently filed and copending application Ser. No. 563,144, now U.S. Pat. No. 4,534,881, entitled "Inhibition of Inorganic or Biological CaCO.sub.3 Deposition by Poly Amino Acid Derivatives", incorporated herein by reference, further disclose a method of inhibiting the formation of inorganic or biological CaCO.sub.3 deposition by applying a synthetic amino acid polymer having a proteinaceous matrix-like structure.
Sikes, C. S. and Wheeler, A. P., in concurrently filed and copending application Ser. No. 563,145 entitled "Inhibition of Inorganic or Biological CaCO.sub.3 Deposition by Synthetic Polysaccharide Derivatives", incorporated herein by reference, further disclose a method of inhibiting the formation of inorganic or biological deposition of CaCO.sub.3 by applying to a surface in contact with CaCO.sub.3 a synthetic saccharide polymer having a polysaccharide-matrix-like structure.
None of the cofiled, copending applications by the present inventors are considered prior art to the present invention.
An interest in further elucidating the role played by the soluble protein matrix from CaCO.sub.3 -forming animals in the inhibition of CaCO.sub.3 encrustration and growth of calcifying organisms, prompted the present inventors to search for other potent and commercially useful inhibitors of said processes. This successful innovation and perfection, for the first time, of the process for the purification of new proteinaceous fractions (or peptide-containing fractions) from the soluble matrix of CaCO.sub.3 -forming animals, resulting in a significantly more potent calcium carbonate-deposition inhibitor, now opens the possibility of using the animal-derived protein fractions for the inhibition of calcium carbonate deposition in pipes, boilers, and the like, of widespread use in industrial environments, as well as for the prevention of fouling of surfaces in marine environments. The use of these highly potent protein-containing inhibitors for the inhibition or retardation of CaCO.sub.3 deposition has heretofor been unknown in the art.